DK2929051T3 - PROCEDURE FOR DETECTING HELICOBACTER PYLORI DNA IN A FAILURE TEST - Google Patents

Description

DESCRIPTION

[0001] The present invention relates to the field of polymerase chain reaction (PCR) based diagnostic assays. More specifically, the present invention provides a PCR based method for detecting chronic gastritis causing bacteria, namely Helicobacter pylori, in a stool sample. The present invention is based on the use of oligonucleotide primers and probes specific to the H. pylori 23S rRNA gene.

BACKGROUND OF THE INVENTION

[0002] H. pylori is a gram-negative and microaerophilic bacterium, which causes chronic gastritis. It is estimated that about half of the world's population are infected with this bacterium. Most of the H. pylori infected individuals are asymptomatic despite gastritis but a minority will develop severe sequelae. H. pylori is the key factor in the development of duodenal or gastric ulcers, and the most common single risk factor for non-cardia gastric cancer. The successful eradication of H. pylori infection leads to healing of peptic ulcer disease (Ford et al, 2003) and in long-term relief of dyspeptic symptoms even in some patients without ulcers (Ford et al., 2004). Eradication therapy of H. pylori infection is also recommended as the first-line treatment for low-grade mucosa-associated lymphoid tissue (MALT) lymphoma and on the basis of epidemiological studies successful H. pylori eradication therapy may lead to a decreased number of gastric cancer cases (Kosunen et al, 2011; Chey and Wong, 2007).

[0003] Diagnostic methods for the detection of H. pylori infection can be divided into invasive (gastroscopy is required) and non-invasive methods. Although several methods give highly accurate results, there is not a single gold standard for the diagnosis of H. pylori infection but the selection of the methods depends on the clinical situation and if there is otherwise a need for gastroscopy. According to the test-and treat strategy, patients with a low risk for gastric cancer can be tested for H. pylori with non-invasive methods and treated if H. pylori is detected.

[0004] Although non-invasive diagnostic methods, such as urea breath tests and stool antigen tests, in general show highly accurate detection rates, these methods do not give any information on the antimicrobial susceptibility of the infecting isolate of H. pylori. Furthermore, even if gastric biopsies are taken in gastroscopy and sent for culture, antimicrobial susceptibility testing results are usually not available in all positive cases due to the low sensitivity of culture. However, the need for antimicrobial susceptibility testing of H. pylori is increasing. Eradication therapy of H. pylori usually consists of two different antimicrobial agents and a proton pump inhibitor. Clarithromycin is an important component of the classical triple therapy and in the case of clarithromycin resistant H. pylori, the clarithromycin-based regimen results in eradication failure in the vast majority of the cases (Fischbach and Evans, 2007).

[0005] Due to the increasing clarithromycin resistance rates (Malfertheiner et al., 2012) the latest European guidelines recommend the use of molecular tests in the detection of H. pylori in gastric biopsies and antimicrobial susceptibility testing if culture is not available. Molecular methods have been developed for the detection of H. pylori and the simultaneous testing for clarithromycin susceptibility of the isolates. Clarithromycin resistance of H. pylori is well known and due to point mutations within the peptidyltransferase region of the 23S rRNA gene.

[0006] The high clinical relevance of H. pylori infection in gastric mucosa has stimulated the development of several PCR based diagnostic methods detecting H. pylori DNA in stool samples. However, the problem of low sensitivity has frequently arisen. These results may have been due to a lack of intact H. pylori DNA in stools. In contrast to intestinal bacterial pathogens, which are found in viable form at high concentrations in stools, living H. pylori is most likely not present at all and, consequently, its DNA may be present only in a degraded form rendering the detection more challenging. Although some PCR-based methods have shown accurate results in the detection of H. pylori and testing of clarithromycin susceptibility in gastric biopsies, problems have arisen when the methods have been used in stool samples. Due to some major limitations, such as PCR inhibitors (Monteneiro, 1997) and low concentrations of mostly fragmented H. pylori DNA in fecal samples, it has been difficult to develop methods sensitive enough. The PCR-based methods have only shown sensitivities about 60% when applied for stool samples (Lottspeich, 2007 etc). The purpose of the present study was to develop a highly accurate non-invasive method for detection of H. pylori and concomitant clarithromycin susceptibility of the isolate in fecal samples.

[0007] Schabereiter-Gurtner et al. (2004) disclosed a real-time PCR assay for detection of H. pylori infection and simultaneous clarithromycin susceptibility testing in stool samples. In practice, the authors detected point mutations in the 23S rRNA gene of H. pylori associated with clarithromycin resistance. However, Lottspeich et al. (2007) evaluated the method and concluded that detection of H. pylori DNA in stool samples by real-time PCR is a difficult task and that this method cannot replace the stool antigen EIA for the accurate diagnosis of H. pylori infection. Later, Scaletsky et al. (2011) found that the method proved to be appropriate for H. pylori clarithromycin susceptibility testing, although the possibility of missing some positive results should be taken into account. Other publications disclosing primers and/or probes specific to H. pylori 23S rRNA gene are: Fontana et al. ,2003; Noguchi et al., 2007; Dewhirst et al., 2005; Maeda et al., 1998; Khan et al., 2004; and Rimbara et al., 2005.

[0008] PCR assays for detection of H. pylori directed to other target genes than the 23S rRNA gene are disclosed in the following publications: Falsafi et al., 2009; Singh et al., 2008; Monteiro et al., 2001; Makristathis et al., 1998; Mishra et al, 2008; and Burucoa et al., 1999.

[0009] In the development of PCR assays, one of the most important factors is to locate oligonucleotide sequences that enable reliable species-specific amplification, detection and quantification. It is of utmost importance that a given set of oligonucleotides, designed to amplify H. pylori, does not cross-react with DNA originating from any other species possibly present in a sample. Finding such sequences can be far from trivial, at least for the following reasons: 1) Many of the species are relatively closely related, making it challenging to locate sequences that are unique for each species; 2) Pathogen strains originating from a single species can be genetically diverged, making it difficult to locate sequences that would enable equally efficient amplification of all strains within a species; 3) The sample may contain PCR inhibitors or as in this case the sample contains mainly fragmented target DNA, since H. pylori typically thrives in gastric mucosa and will very likely die and deteriorate in large intestine. Hence, effective amplification of pathogen DNA from a stool sample requires oligonucleotide design enabling high PCR efficiency (optimally as close to 100% as possible).

[0010] Compared to the prior art, the present invention provides at least the following major advantages: the difference of Tm of outer and inner primers is optimized to achieve simpler reaction routines, e.g. the nested PCR reaction of the present invention can be performed in a single vessel. Rimbara et al, 2005, and Noguchi et al., 2007, disclose methods where nested PCR is performed sequentially in two separate reactions. The robustness of the PCR reaction of the present invention is also on the level that there is no need to isolate and purify the DNA from a stool sample by phenol extraction or sample homogenization as is done in Rimbara et al, 2005, and Noguchi et al., 2007, respectively. Furthermore, the length of the amplicon which is amplified in the second reaction of the nested PCR in Rimbara et al, 2005, and Noguchi et al., 2007, is too long to be sensitively detected in real-time PCR. The length of the amplicon amplified by the inner primers of the present invention is 143 bp while in Rimbara it is 463 bp and in Noguchi it is 367 bp.

[0011] The disclosures by Fontana et al., 2003, JP 2005168474, and Booka et al., 2005 are not directed to a nested PCR reaction and consequently the primers disclosed are not compatible with nested reactions without modification. Further, the length of the amplicon amplified in Fontana et al., 2003, is 991 bp and is thus too long to be detected in real time PCR. It is also noteworthy that Fontana et al. are amplifying a different region of 23S rRNA gene than Noguchi et al. Therefore, it is clear that the prior art is teaching that there are alternative regions in 23S rRNA which are suitable as target regions for a PCR based detection of the presence of Helicobacter strains. In JP 2005168474, the primers seem not to be specific to Helicobacter but may also cross-react with Campylobacter strains.

[0012] Although numerous PCR based assays for detecting H. pylori are already disclosed, there is still a need in the field for a PCR assay which is able to provide high specificity and reliability for the detection. The present inventors have now located DNA sequence regions in H. pylori 23S rRNA gene that are surprisingly well-suited for specific and sensitive amplification of H. pylori DNA from stool. Optimal primers and quantitative PCR probes have been designed and validated for identification of the presence of H. pylori in patients.

SUMMARY OF THE INVENTION

[0013] The invention is as set out in the claims. The present dislcosure provides a method of detecting H. pylori DNA in a stool sample, the method comprising the steps of: 1. a) performing a PCR reaction comprising DNA isolated from a stool sample as a template and an oligonucleotide primer set specific for amplifying an H. pylori-specific target sequence in an H. pylori 23S rRNAgene in an amplification reaction, wherein the oligonucleotide primer set comprises an oligonucleotide consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 1 and an oligonucleotide consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 2; and 2. b) detecting H. pylori DNA in said stool sample when the H. pylori -specific target sequence is amplified.

[0014] The present disclosure also provides an oligonucleotide primer set comprising an oligonucleotide consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 1 and an oligonucleotide consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 2, wherein the oligonucleotide primer set amplifies a target sequence in the 23S rRNA gene of H. pylori.

[0015] The present disclosure is further directed to a kit for detecting H. pylori DNA in a stool sample, the kit comprising the oligonucleotide primer set as described above; and a reagent for performing amplification of a nucleic acid in a PCR reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]

Figure 1. Positive H. pylori result with a conserved region probe (JOE) and positive result for the presence of clarithromycin resistance mutation (ROX). In addition, internal control is positive. Wild type probe (FAM) detecting clarithromycin susceptibility genotype is negative.

Figure 2. Positive H. pylori result with a conserved region probe (JOE) and positive result for the presence of wild type probe (FAM) excluding clarithromycin resistance mutation. In addition, internal control (CY5) is positive. Resistance mutation probes detecting clarithromycin resistance (ROX) are negative. See Figure 2 for results.

Figure 3. Negative result for H. pylori: only internal control is positive, no PCR inhibition detected. See Figure 3 for results.

DETAILED DESCRIPTION OF THE INVENTION

[0017] As used herein, a "target sequence" present in a nucleic acid sample is a strand of H. pylori DNAto be primed and extended by a "primer". A target sequence may be either single-stranded or in a duplex with its complementary sequence. In certain embodiments, a target sequence may not be present in a nucleic acid sample, but may be present later as a result of transcription from another nucleic acid present in said nucleic acid sample. Target sequences may be from any number of sources based on the purpose of the assay being carried out. Target sequence of the present invention is purified to some degree prior to the amplification reactions described herein.

[0018] As used herein, the term "oligonucleotide" refers to any polymer of two or more of nucleotides, nucleosides, nucleobases or related compounds used as a reagent in the DNA amplification methods of the present invention. The oligonucleotide may be DNA and/or RNA and/or analogs thereof. The term oligonucleotide does not denote any particular function to the reagent; rather, it is used generically to cover all such reagents described herein. Specific oligonucleotides of the present invention are described in more detail below. As used herein, an oligonucleotide can be virtually any length, limited only by its specific function in the DNA amplification reaction. Oligonucleotides of a defined sequence and chemical structure may be produced by techniques known to those of ordinary skill in the art, such as by chemical or biochemical synthesis, and by in vitro or in vivo expression from recombinant nucleic acid molecules, e.g., bacterial or viral vectors. Oligonucleotides may be modified in anyway, as long as a given modification is compatible with the desired function of a given oligonucleotide. One of ordinary skill in the art can easily determine whether a given modification is suitable or desired for any given oligonucleotide of the present invention. Modifications include, but are not limited to base modifications, sugar modifications or backbone modifications. While design and sequence of oligonucleotides for the present invention depend on their function as described below, several variables must generally be taken into account. Among the most critical are: length, G/C content, melting temperature (Tm), Gibb free energy (G), specificity, self-complementarity and complementarity with other oligonucleotides in the system, polypyrimidine (T, C) or polypurine (A, G) stretches, and the 3'-end sequence. Controlling for these and other variables is a standard and well-known aspect of oligonucleotide design, and various computer programs are readily available to screen large numbers of potential oligonucleotides for optimal ones.

[0019] As used herein, the term "PCR amplifying" or "PCR amplification" refers generally to cycling polymerase-mediated exponential amplification of nucleic acids employing primers that hybridize to complementary strands, as described for example in Innis et al, PCR Protocols: A Guide to Methods and Applications, Academic Press (1990). Devices have been developed that can perform thermal cycling reactions with compositions containing fluorescent indicators which are able to emit a light beam of a specified wavelength, read the intensity of the fluorescent dye, and display the intensity of fluorescence after each cycle. The amplification product contains a sequence having sequence identity with a target nucleic acid sequence or its complement and can be detected with, for example, an intercalating dye or a detection probe having specificity for a region of the target nucleic acid sequence or its complement. The PCR reaction of the present invention is preferably performed as a real-time PCR assay. As used herein, the term "probe" refers to any of a variety of signaling molecules indicative of amplification. For example, SYBR® Green and other DNA-binding dyes are detector probes. Some detector probes can be sequence-based, for example 5' nuclease probes. Various detector probes are known in the art, for example TaqMan® probes (See U.S. Patent No. 5,538,848). The melting temperature, Tm, of the probes can be increased by addition of modified nucleotides. The amount of modified nucleotides in one probe is 1, 2, 3, 4 or more. The modified nucleotide can be a LNA nucleotide (Exiqon A/S), minor groove binder (MGB™), SuperBase, or Peptide Nucleic Acid (PNA) or any other modification increasing the Tm of the probe.

[0020] H. pylori DNA is present only in low level in feces, since H. pylori is an upper intestinal track pathogen. Therefore, the right choice of highly specific and sensitive primers is of crucial importance in order to obtain accurate results from a PCR based assay using DNA template isolated from a stool sample. The present invention provides a method and an oligonucleotide primer set for amplifying at least one target sequence of the 23S rRNA gene of H. pylori. The developed assay can detect H. pylori infection with high sensitivity and simultaneously evaluate clarithromycin resistance. The effect of the invention is particularly related to the choice of target sites for the outer primers, i.e. SEQ ID NOS: 1 and 2.

[0021] Accordingly, the present disclosure is directed to a method of detecting H. pylori DNA in a stool sample, the method comprising the steps of: 1. a) performing a PCR reaction comprising DNA isolated from a stool sample as a template and an oligonucleotide primer set specific for amplifying an H. pylori-specific target sequence in an H. pylori 23S rRNA gene in an amplification reaction, wherein the oligonucleotide primer set comprises an oligonucleotide consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 1 and an oligonucleotide consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 2; and 2. b) detecting H. pylori DNA in said stool sample when the H. pylori -specific target sequence is amplified in step a).

Since nested PCR generally increases sensitivity, said PCR reaction is preferably a nested PCR reaction. Accordingly, a second oligonucleotide primer set can be used in said PCR reaction comprising an oligonucleotide consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 3 and an oligonucleotide consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 4. Said second oligonucleotide primer set is directed to the amplification of sites of point mutations associated with clarithromycin resistance of certain H. pylori strains. The method is thus also further directed to the detection of the mutations in H. pylori 23S rRNA gene associated with clarithromycin resistance in H. pylori. The presence of said mutations can be detected by a probe or probes detecting the presence of mutations in said gene, preferably at positions corresponding to positions 2514 and/or 2512 of SEQ ID NO: 13 (GenBank: U27270.1). Preferably, said mutations are detected by a probe consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 7 and/or a probe consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 8. Further, for the detection of said mutations a probe or probes consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 5 and/or 6 or comprising or consisting of the sequence as set forth in SEQ ID NO: 5 and/or 6 can be used.

[0022] One of the most preferred embodiments is to perform steps a) and b) of the method in a single vessel (e.g. as described by Strauss et al., 2000). This approach lowers the risk of contamination. To achieve this goal, the melting temperatures of the first and second oligonucleotide primer set must be designed so that the temperature difference between the sets is at least 3 degrees centigrade, preferably 3.5 or 4 degrees centigrade.

The target sequence in H. pylori 23S rRNA gene for the first oligonucleotide primer set consisting of SEQ ID NOS: 1 and 2 corresponds to positions 1937-2793 of SEQ ID NO: 13.

The target sequence in H. pylori 23S rRNA gene for the second oligonucleotide primer set consisting of SEQ ID NOS: 3 and 4 corresponds to positions 2482-2624 of SEQ ID NO: 13.

[0023] Preferably, the detection of H. pylori DNA in the stool sample when the H. pylori-specific target sequence is amplified is performed using a DNA chip, gel electrophoresis, a radiation measurement, a fluorescence measurement, or a phosphorescence measurement. A person skilled in the art may use the primers and probes of the disclosure also in other methods and platforms utilizing PCR.

[0024] The present disclosure also provides an oligonucleotide primer set comprising an oligonucleotide consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 1 and an oligonucleotide consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 2, wherein the oligonucleotide primer set amplifies a target sequence in a 23S rRNA gene of H. pylori. Preferably, the oligonucleotide primer set comprises or consists of the nucleotide sequence as set forth in SEQ ID NO: 1 and the nucleotide sequence as set forth in SEQ ID NO: 2.

Preferably, the oligonucleotide primer set further comprises an oligonucleotide consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 3 and an oligonucleotide consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 4. More preferably, said oligonucleotide primer set comprises or consists of the nucleotide sequence as set forth in SEQ ID NO: 3 and the nucleotide sequence as set forth in SEQ ID NO: 4.

The oligonucleotide primer set may further comprise a probe consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 7 and/or a probe consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 8. More preferably, said oligonucleotide primer set comprises or consists of the nucleotide sequence as set forth in SEQ ID NO: 7 and the nucleotide sequence as set forth in SEQ ID NO: 8. Most preferably, said oligonucleotide primer set further comprises or consists of the nucleotide sequence as set forth in SEQ ID NO: 5 and the nucleotide sequence as set forth in SEQ ID NO: 6.

[0025] The present disclosure is also providing a kit for detecting H. pylori DNA in a stool sample, the kit comprising at least one of the oligonucleotide primer sets as described above; and a reagent for performing amplification of a nucleic acid in a PCR reaction. Preferably, said reagent is selected from a group consisting of DNA polymerase, dNTPs, and a buffer.

EXPERIMENTAL SECTION

[0026] The aim in the design phase was to develop a highly H. pylori specific PCR that would not amplify other closely related species. The new assay is developed and optimized using sequencing confirmed H. pylori strains with and without clarithromycin resistance mutations. Then analytical specificity was tested against 50 clinically relevant pathogenic and non-pathogenic bacterial species, including both gram-negative and gram-positive species. No significant cross-reactivity was detected. The analytical sensitivity with 95% confidence for positive replicas was 10fg, which corresponds to two genomic copies or one bacterium. The clinical sensitivity and specificity were analysed using gastric biopsy, histology and Helicobacter culture with antimicrobial susceptibility testing as a golden standard reference method. Correct identification was achieved in 94% of samples and a correct antimicrobial susceptibility in 90% of a total 80 patient samples studied. As a control, a template from Oryza sativa, terminal flower gene Ory, was used.

Nucleid acid extraction [0027] The total nucleid acids were extracted using bioMerieux NucliSens Kits, and semi-automated easyMAG instrument for extraction. Both generic and specific B protocol was successfully tested. The specific B protocol was slightly better in qPCR performance, and it was selected for all experiments. A loopful of stool, app. 10% solution, was inoculated into 2mL of the kit lysis buffer, mixed rigorously for 5s and incubated at least 15min in room temperature according to manufacturer's instruction. The extraction volume was 25μΙ. PCR setup and oligonucleotides [0028] The following PCR program and set of oligonucleotides were utilized for all assays performed with Stratagene MxPro 3005P: 1.95°C 13min 2. 94°C 60 3. 68°C 90s 4. 2-3 x5 cycles 5. 94°C 30s 6. 68°C 60s 7. 2-3 x 15 cycles 8. 94°C 25 s 9. 64°C 25 s 10.8-9x40

Primers

Outer primers: [0029]

Inner primers: [0030]

Control: [0031]

Probes [0032]

[0033] The underlined nucleotides in the above list are LNA nucleotides (Exiqon A/S) increasing the Tm of the probe.

Synthetic Ory template [0034] TGCTCCTAATCCCAGCAACCCAAeeTTGAGGGAATACCTGCACTGGTAAGCTA TGCTCTTGCAATTGTTGTGATTTCTATCATATGTCTCACATTGATTAGTGATCTA (SF.Q ID NO: 12)

Reaction mix

[0035] After initial denaturation and polymerase activation 5 cycles of long extension time was utilized to improve amplification efficacy from less optimal genomic DNA. After generating the few initial copies, the long specific amplification phase was continued 15 more cycles, but no more to maximize amplification efficacy in the following steps (i.e. 8.-10.). Surprisingly, a longer first phase than 20 (5+15) cycles decreased sensitivity and caused a risk for too early amplification in the clarithromycin resistance mutation detection phase leading to great problems with data analysis. The annealing temperature was carefully optimized similar for all probes to be able to detect of single nucleotide mutation. It was difficult to design high sensitivity probes due to rather low melting temperature requirement, which was necessary to avoid putative unintended probe binding in the earlier high temperature specific amplification phase.

Example 1.

[0036] Positive H. pylori result with a conserved region probe (JOE) and positive result for the presence of clarithromycin resistance mutation (ROX). In addition, internal control is positive. Wild type probe (FAM) detecting clarithromycin susceptibility is negative. See Figure 1 for results.

[0037] FAM (green): wild type genotype within clarithromycin mutation hot spot. When positive, no resistance mutation present. Treatment with clarithromycin based combination likely successful.

[0038] JOE (yellow): highly conserved region in the lagging strand, indicates presence of H. pylori in the sample regardless resistance mutations present.

Note! If a new, or a double mutation appears, the JOE is the only positive probe in addition to internal control = clarithromycin resistant genotype present.

[0039] ROX (red): a mutation in A2142G or A2143G position linked to clarithromycin resistance. The most common types detected.

[0040] CY5 (far red): inhibition control.

Example 2.

[0041] Positive H. pylori result with a conserved region probe (JOE) and positive result for the presence of wild type probe (FAM) excluding clarithromycin resistance mutation. In addition, internal control (CY5) is positive. Resistance mutation probes detecting clarithromycin resistance (ROX) are negative. See Figure 2 for results.

Example 3.

[0042] Negative result for H. pylori: only internal control is positive, no PCR inhibition detected. See Figure 3 for results.

REFERENCES

[0043]

Booka etal., 2005, Helicobacter, 10:205-213

Burucoa etal., 1999, Journal Of Clinical Microbiology, 37(12):4071-4080

Chey and Wong, 2007, Am J Gastroenterol, 102:1808-1825

Dewhirst et al., 2005, Journal Of Bacteriology, 187(17):6106-6118

Falsafi et al., 2009, World J Gastroenterol, 15(4): 484-488

Fischbach and Evans, 2007, Aliment Pharmacol Ther 2007;26:343e57

Fontana etal.,2003, Journal Of Clinical Microbiology, 41(8):3636-3640

Ford et al., 2003, The Cochrane Database of Systematic Reviews 2003, Issue 4.

Ford et al., 2004, Am J Gastroenterol, 99:1833-1855

Khan et al., 2004, Antimicrobial Agents And Chemotherapy, 48(9):3567-3569

Kosunen etal., 2011, Int. J. Cancer: 128:433-439

Lottspeich, 2007, Journal Of Clinical Microbiology, 45(6):1718-1722

Maeda et al., 1998, Gut, 43:317-321

Makristathis et al., 1998, Journal Of Clinical Microbiology, 36(9):2772-2774

Malfertheiner et al., 2012, Gut 61:646e664

Mishra et al, 2008, J Infect Developing Countries. 2(3): 206-210

Monteiro et al., 2001, Journal of Microbiological Methods. 45:89-94

Noguchi et al., 2007, Journal of Medical Microbiology, 56, 1174-1180

Rimbara et al., 2005, Current Microbiology, 51:1-5

Scaletsky et al.,2011, Helicobacter, 16: 311-315

Schabereiter-Gurtner et al., 2004, Journal Of Clinical Microbiology, 42(10):4512-4518 Singh et al., 2008, Helicobacter, 13(1): 30-34

Strauss et al., 2000, Diagnostic Molecular Pathology 9(3): 151-157, 2000 SEQUENCE LISTING

[0044] <110> Amplidiag Oy <120> Method for detecting Helicobacter pylori DNA in a stool sample <130> 52442 <150> FI 20126271 <151 > 2012-12-05 <160> 13 <170> Patentln version 3.1

<210> 1 <211> 30 <212> DNA <213> Helicobacter pylori <400> 1 gctagtctaa gggcgtagat tggagggaag 30

<210> 2 <211> 31 <212> DNA <213> Helicobacter pylori <400>2 gcttgtgcca ttacactcaa cttgcgattt c 31

<210>3 <211> 19 <212> DNA <213> Helicobacter pylori <400>3 ggtgaaaatt cctcctacc 19 <210> 4 <211 > 18

<212> DNA <213> Helicobacter pylori <400>4 caaggatggc tccataag 18

<210>5 <211 > 16 <212> DNA <213> Helicobacter pylori <400>5 caagacggaa agaccc 16

<210>6 <211 >23 <212> DNA <213> Helicobacter pylori <400>6 caaagcctcc cacctatcct gcg 23

<210>7 <211 > 16 <212> DNA <213> Helicobacter pylori <400>7 caagacggag agaccc 16

<210>8 <211 > 16 <212> DNA <213> Helicobacter pylori <400>8 caagacggga agaccc 16 <210>9 <211 >20 <212> DNA <213> Oryza sativa <400>9 ctaatcccag caacccaacc 20 <210> 10 <211 > 30 <212> DNA <213> Oryza sativa <400> 10 ctaatcaatg tgagacatat gatagaaatc 30 <210> 11 <211> 19 <212> DNA <213> Oryza sativa <400> 11 cctgcactgg taagctatg 19 <210> 12 <211> 108 <212> DNA <213> Oryza sativa <400> 12 tgctcctaat cccagcaacc caaccttgag ggaatacctg cactggtaag ctatgctctt 60 gcaattgttg tgatttctat catatgtctc acattgatta gtgatcta 108

<210> 13 <211 >3857 <212> DNA <213> Helicobacter pylori <400> 13 aaagcttcat ccacccccco catcccatca tttccaatca cttttatcca tttctttcaa 60 acccaaaaac tttaagcaaa ctttaagcat gtctataatt acatttogtt ttaaagacaa 120 gctttaaaag tctttaattg aaccactcaa acaagttcta caagctaaag ctttaaataa 180 aacecaccag ctggtaaaac ttgagtgtta taaaaagatt agggatcaag oatttttagt 240 cttcttfeaag ggtttaacat taagagtgat tatagcaagt ttttaaagaa aaacgaagtt 300 atttgattta acattgttaa tagcctatgt aaaagtaaag taaaactaca ataactotgt 360 cttatattca ttaaggcagt ggtagcgctg aagaatgttc gtgcaattgt cgttattcat 420 tataaaaggg cgggttttaa aggatatttt aaaatttaaa acaagctttt aagagcagat 480 ggcggatgcc ttgccaaaga gaggcgatga aggacgtact agactgcgat aagctatgcg 540 gagctgtcaa ggagctttga tgcgtagatg tccgaatggg gcaacccaac taatagagat 600 attagttact ctaacagaga gcgaacctag tgaagtgaaa catctcagta actagaggaa 660 aagaaatcaa cgagattccc taagtagtgg cgagcgaacg gggaaaaggg caaaccgacrt 720 gcttgcattc ggggttgagg actgcaacat ccaagagaac gctttagcag agttacctgg 780 aaaggtaagc catagaaagt gatagccttg tatgcgacaa ggcgttttta ggtagcagta 840 tccagagtag gccaggacac gaggaatcca ggttgaagcc ggggagacca ctctccaact 900 ctaaatacta ctctttgagc gatagcgaac aagtaccgtg agggaaaggt gaaaagaacc 960 gcagtgagcg gagtgaaata gaacctgaaa ccatctgctt acaatcattc agagccctat 1020 gatttatcag ggtgatggac tgccttttgc ataatgatcc tgcgagttgt ggtatctggc 1080 aaggttaagc gaatgcgaag ccgtagcgaa acgagttctt aatagggcga acaagtcaga 1140 tgctgcagac ccgaagcfcaa gtgatctatc catggccaag ttgaaacgcg tgtaatagcg 1200 cgtggaggac tgaactccta cccattgaaa cgggttggga tgagctgtgg ataggggtga 1260 aaggccaaac aaacttagtg atagctggtt ctcttcgaaa tatatttagg tatagcctca 1320 agtgataata aaagggggta gagcgctgat tgggctaggg ctgctcgccg cggtaccaaa 1380 ccctatcaaa cttcgaatac cttttatcgt atcttgggag tcaggcggtg ggtgataaaa 1440 tcaatcgtca aaaggggaac aacccagact accaaataag gtccctaagt tctattctga 1500 gtggaaaaag atgtgtggct acfcaaaacaa ccaggaggtt ggcttagaag cagccatcct 1560 ttaaagaaag cgtaacagct cactggtcta gtggtcatgc gctgaaaata taacggggct 1620 aagatagaca ccgaatttgt agattgtgtt aaacacagtg gtagaagagc gttcatacca 1680 gcgttgaagg tataccggta aggagtgctg gagcggtatg aagtgagcat gcaggaatga 1740 gtaacgataa gatatatgag aattgtatcc gccgtaaatc taaggtttcc tacgcgatgg 1800 tcgtcatcgt agggLLaqLc qqqLccLaaq ccqaqLccga aaqqqgtagg tgatggcaaa 1860 ttggttaata ttccaatacc gactgtggag cgtgatgggg ggacgcatag ggttaagcga 1920 gctagctgat ggaagcgcta gtctaagggc gtagattgga gggaaggcaa atccacctct 1980 gtat-tgaaa cccaaacagg ctctttgagt ccttttagga caaagggaga atcgctgata 2040 ccgtcgtgcc aagaaaagoc tctaagcata tccatagtcg tccgtaccgc aaaccqacac 2100 aggtagatga gatgagtatt ctaaggcgcg tgaaagaact ctggttaagg aactctgcaa 2160 actagcaegg taagttcgcg ataaggtgtg ccacagcgat gtggtctcag caaagagtcc 2220 ctcccgactg tttaccaaaa acacagcact ttgccaactc gtaagaggaa gtataaggtg 2280 tgacgcctgc ccggtgctcg aagqttaaga ggatgcgtca gtcgcaagat gaagcgttga 2340 attgaagccc gagtaaacgg cggccgtaac tataacggtc craaggtagc gaaattcctt 2400 gtcggttaaa tacogaoctg catgaatggc gtaacgagat gggagctgta tcaaccagag 2460 attcagtgaa attgtagtgg aggtgaaaat: tcctcctacc cgcggcaaga cggaaagacc 2520 ccgtggacct ttactacaac ttagcactgc taafcgggaat atcatgcgca ggataggtgg 2580 gaggctttga agtaaggget ttggctctta tggagtcatc cttgagatac cacccttgat 2640 gtttcfcgtta gctaaGtggc! ctgtgttatc cacaggcagg acaatgcttg gtgggtagtt 2700 tgactggggc ggtcgctcct aaaaagtaac ggaggcttgc aaaggttggc tcattgGggt 2760 tggaaatcgc aagttgagtg taatggcaca agccagcctg actgtaagac atacaagtca 2820 agcagagaog aaagtcggtc atagtgatcc ggtggttctg tgtggaaggg ccatcgctca 2880 aaggataaaa ggtacoccgg ggataacagg ctgatctccc ccaagagctc acatcgacgg 2940 ggaggtttgg caoctcgatq tcggctcatc goafcccfcggg gctggagcag gtcccaaggg 3000 tatggctgtt cgccatttaa agcggtacgc gagctgggtt cagaacgtcg tgagacagtt 3060 cggtccctat etgccgtggg cgtaggaaag ttgaggagag ctgtceGtag tacgagagga 3120 L-^ WW^Waa ^α^υαυυ^υι· yyy L-ayL-OL-a _> j- u v cacggatgtg ataactgctg aaagpatota agcaggaacc aactccaaga taaactttcc 3240 ctgaagctcg pacaaagact atgtgcttga tagggtagat gtgtgagcgc agtaatgcgt 3300 ttagctgaot actactaata gagcgtttgg cttgtttttt gctttttgat aagataacgg 3360 caataagcgc gaatgggtta ccacfcgcctt actgagtgta agagagttgg agttttatga 3420 agacttttat aagattaaac tttaatgagg aatgagatac catctcaatg gtttaaagtt 3480 aaaggctatt aacgatcttc tttgttaaaa acagct.cccc tataaagaga aaggggagtt 3540 aagggtaaat gcgtttttat ctttagctcc cttttccttg tgcGtttaga gaagaggaac 3600 tacccagtta accattccga acctggaagt caagctcttc atcgctgata ataotgctot 3560 tttcaagagt gggaatgtag gtcggtgcag ggatagggaa atgttttttt agtcttgctt 3720 ttttattfcga tttcattatt gactcattgt tttgtttgtt taggtggttt attggggttt 3780 ggttgttttg ttgatttagt tttcatgctc taaaccgatg aaaggttgtt tgaagtcttc 3840 tctgttcata aacttgc 3857

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

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Patentkrav 1. Fremgangsmåde til at detektere Helicobacter pylori-DNA i en afføringsprøve, hvilken fremgangsmåde omfatter: a) at udføre en nested PCR-reaktion omfattende DNA isoleret fra en afføringsprøve som en template og et første og andet oligonukleotid-primersæt specifikt for amplificering af H. pylori-specifikke målsekvenser i et H. pylori 23S rRNA-gen i en amplifikationsreaktion, hvor det første oligonukleotid-primersæt omfatter en oligonukleotid-primer omfattende nukleotidsekvensen som fremsat i SEQ ID NO: 1 eller bestående af mindst 10 sammenhængende nukleotider til stede i nukleotidsekvensen som fremsat i SEQ ID NO: 1 og en oligonukleotid-primer omfattende nukleotidsekvensen som fremsat i SEQ ID NO: 2 eller bestående af mindst 10 sammenhængende nukleotider til stede i nukleotidsekvensen som fremsat i SEQ ID NO: 2, hvor det andet oligonukleotid-primersæt omfatter en oligonukleotid-primer omfattende nukleotidsekvensen som fremsat i SEQ ID NO: 3 eller bestående af mindst 10 sammenhængende nukleotider til stede i nukleotidsekvensen som fremsat i SEQ ID NO: 3 og en oligonukleotid-primer omfattende nukleotidsekvensen som fremsat i SEQ ID NO: 4 eller bestående af mindst 10 sammenhængende nukleotider til stede i nukleotidsekvensen som fremsat i SEQ ID NO: 4, hvor den H. pylori-specifikke målsekvens af det første oligonukleotid-primersæt er en nukleotidregion af et H. pylori 23S rRNA-gen svarende til positionerne 1937-2793 af SEQ ID NO: 13 og mindst en del af nævnte nukleotidregion amplificeres i PCR-reaktionen med det første oligonukleotid-primersæt, og hvor den H. pylori-specifikke målsekvens af det andet oligonukleotid-primersæt er en nukleotidregion af et H. pylori 23S rRNA-gen svarende til positionerne 2482-2624 af SEQ ID NO: 13 og mindst en del af nævnte nukleotidregion amplificeres i PCR-reaktionen med det andet oligonukleotid-primersæt b) at detektere H. pylori-DNA i nævnte afføringsprøve, idet de H. pylori-specifikke målsekvenser amplificeres. 2. Fremgangsmåden ifølge krav 1, hvor mutationerne i H. pylori 23S rRNA-gen associeret med clarithromycin-resistens i H. pylori yderligere detekteres af en første probe bestående af mindst 10 sammenhængende nukleotider til stede i en nukleotidsekvens som fremsat i SEQ ID NO: 7 og en anden probe bestående af mindst 10 sammenhængende nukleotider til stede i en nukleotidsekvens som fremsat i SEQ ID NO: 8 eller hvor nævnte første probe omfatter nukleotidsekvensen som fremsat i SEQ ID NO: 7 og nævnte anden probe omfatter nukleotidsekvensen som fremsat i SEQ ID NO: 8. 3. Fremgangsmåden ifølge krav 1 eller 2, hvor detektionen af H. py/or/-DNA i afføringsprøven, når de H. pylori-specifikke målsekvenser er amplificerede, udføres under anvendelse af en DNA-chip, gelelektroforese, en strålingsmåling, en fluorescensmåling, eller en phosphorescensmåling. 4. Fremgangsmåden ifølge et hvilket som helst af kravene 1-3, hvor trinnene a) og b) udføres i en enkelt beholder. 5. Fremgangsmåden ifølge et hvilket som helst af de foregående krav, hvor nævnte fremgangsmåde udføres som et realtime-PCR-assay. 6. Oligonukleotid-primersæt omfattende et oligonukleotid bestående af mindst 10 sammenhængende nukleotider til stede i en nukleotidsekvens som fremsat i SEQ ID NO: 1 eller omfattende nukleotidsekvensen som fremsat i SEQ ID NO: 1, et oligonukleotid bestående af mindst 10 sammenhængende nukleotider til stede i en nukleotidsekvens som fremsat i SEQ ID NO: 2 eller omfattende nukleotidsekvensen som fremsat i SEQ ID NO:2, et oligonukleotid bestående af mindst 10 sammenhængende nukleotider til stede i en nukleotidsekvens som fremsat i SEQ ID NO: 3 eller omfattende nukleotidsekvensen som fremsat i SEQ ID NO:3, og et oligonukleotid bestående af mindst 10 sammenhængende

Claims (3)

nukleotider til stede i en nukleotidsekvens som fremsat i SEQ ID NO: 4 eller omfattende nukleotidsekvensen som fremsat i SEQ ID NO:4, hvor oligonukleotid-primersættet amplificerer en målsekvens i et 23S rRNA-gen af H. pylori.nucleotides present in a nucleotide sequence set forth in SEQ ID NO: 4 or comprising the nucleotide sequence set forth in SEQ ID NO: 4, wherein the oligonucleotide primer set amplifies a target sequence in a 23S rRNA gene of H. pylori. 7. Oligonukleotid-primersættet ifølge krav 6, yderligere omfattende en probe bestående af mindst 10 sammenhængende nukleotider til stede i en nukleotidsekvens som fremsat i SEQ ID NO: 7 og en probe bestående af mindst 10 sammenhængende nukleotider til stede i en nukleotidsekvens som fremsat i SEQ ID NO: 8.The oligonucleotide primer set of claim 6, further comprising a probe consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 7 and a probe consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ. ID NO: 8. 8. Kit til at detektere H. pylori-DNA i en afføringsprøve, hvilket kit omfatter: oligonukleotid-primersættet ifølge krav 6 eller 7; og en reagens til at udføre amplifikation af en nukleinsyre i en PCR-reaktion, hvor reagensen er valgt fra gruppen bestående af: DNA-polymerase, dNTP’er, og en buffer.A kit for detecting H. pylori DNA in a stool sample comprising: the oligonucleotide primer set of claim 6 or 7; and a reagent for performing amplification of a nucleic acid in a PCR reaction wherein the reagent is selected from the group consisting of: DNA polymerase, dNTPs, and a buffer.